Prion protein standard and method of making the same

ABSTRACT

The invention provides prion protein standards for use as reference materials for prion detection. The standard may be species specific, i.e. the standard is comprised of a preparation for detection of a single strain prion or it may be prepared to allow detection of multiple prion strains simultaneously. The invention also provides methods of preparing the prion protein standards using a group of non-human host mammals which have their genome manipulated with respect to genetic material related to a PrP gene such that the mammals are susceptible to infection with a prion which generally only infects an animal which is genetically diverse from the host.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase of PCT application no.PCT/US99/27452, filed Nov. 17, 1999 which is a continuation of earlierfiled application Ser. No. 09/199,523, filed Nov. 25, 1998 now issuedU.S. Pat. No. 6,020,537 which is a continuation-in-part of our earlierfiled application Ser. No. 08/935,363, filed Sep. 22, 1997 now issuedU.S. Pat. No. 6,008,435 which is a continuation-in-part of our earlierfiled application Ser. No. 08/692,892, filed Jul. 30, 1996 now issuedU.S. Pat. No. 5,792,901 which is a continuation-in-part of our earlierfiled application Ser. No. 08/521,992, filed Aug. 31, 1995 now issuedU.S. Pat. No. 5,908,969 which is a continuation-in-part of our earlierfiled application Ser. No. 08/509,261, filed Jul. 31, 1995 now issuedU.S. Pat. No. 5,763,740 which is a continuation-in-part of our earlierfiled application Ser. No. 08/242,188, filed May 13, 1994 now issuedU.S. Pat. No. 5,565,186 to which we claim priority and which areincorporated herein by reference in their entirety.

GOVERNMENT RIGHTS

The United States Government may have certain rights in this applicationpursuant to Grant No. NS14069, AG02132, NS22786, AG08967 and AG10770awarded by the National Institutes of Health.

FIELD OF THE INVENTION

This invention relates to the field of bioassays and more particularlyto standards for assays for isolating and detecting a diseaseconformation of a protein present in a sample also containing anon-disease conformation of the protein, and method of making suchstandards.

BACKGROUND OF THE INVENTION

Prions are infectious pathogens that cause invariably fatal priondiseases (spongiform encephalopathies) of the central nervous system inhumans and animals. Prions differ significantly from bacteria, virusesand viroids. The dominating hypothesis is that no nucleic acid isnecessary to allow for the infectivity of a prion protein to proceed.

A major step in the study of prions and the diseases they cause was thediscovery and purification of a protein designated prion protein[Bolton, McKinley et al. (1982) Science 218:1309-1311; Prusiner, Boltonet al. (1982) Biochemistry 21:6942-6950; McKinley, Bolton et al. (1983)Cell 35:57-621. Complete prion protein-encoding genes have since beencloned, sequenced and expressed in transgenic animals. PrP^(C) isencoded by a single-copy host gene [Basler, Oesch et al. (1986) Cell46:417-428 and when PrP^(C) is expressed it is generally found on theouter surface of neurons. Many lines of evidence indicate that priondiseases result from the transformation of the normal form of prionprotein (PrP^(C)) into the abnormal form (PrP^(Sc)). There is nodetectable difference in the amino acid sequence of the two forms.However, PrP^(Sc) when compared with PrP^(C) has a conformation withhigher β-sheet and lower α-helix content [Pan, Baldwin et al. (1993)Proc Natl Acad Sci USA 90:10962-10966; Safar, Roller et al. (1993) JBiol Chem 268:20276-20284]. The presence of the abnormal PrP^(Sc) formin the brains of infected humans or animals is the only disease-specificdiagnostic marker of prion diseases.

PrP^(Sc) plays a key role in both transmission and pathogenesis of priondiseases (spongiform encephalopathies) and it is a critical factor inneuronal degeneration [Prusiner (1997) The Molecular and Genetic Basisof Neurological Disease, 2nd Edition 103-143). The most common priondiseases in animals are scrapie of sheep and goats and bovine spongiformencephalopathy (BSE) of cattle [Wilesmith and Wells (1991) Curr TopMicrobiol Immunol 172:21-38). Four prion diseases of humans have beenidentified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Straussler-Sheinker Disease (GSS), and (4) fatal familialinsomnia (FFI) [Gajdusek (1977) Science 197:943-960; Medori, Tritschleret al. (1992) N Engl J Med 326:444-449]. Initially, the presentation ofthe inherited human prion diseases posed a conundrum which has sincebeen explained by the cellular genetic origin of PrP.

Prions exist in multiple isolates (strains) with distinct biologicalcharacteristics when these different strains infect in geneticallyidentical hosts [Prusiner (1997) The Molecular and Genetic Basis ofNeurological Disease, 2nd Edition:165-186). The strains differ byincubation time, by topology of accumulation of PrP^(Sc) protein, and insome cases also by distribution and characteristics of brain pathology[DeArmond and Prusiner (1997) Greenfield's Neuropathology, 6thEdition:235-280]. Because PrP^(Sc) is the major and very probably theonly component of prions, the existence of prion strains has posed aconundrum as to how biological information can be enciphered in amolecule other than one comprised of nucleic acids. The partialproteolytic treatment of brain homogenates containing some prionisolates has been found to generate peptides with slightly differentelectrophoretic mobilities [Bessen and Marsh (1992) J Virol66:2096-2101; Bessen and Marsh (1992) J Gen Virol 73:329-334; Telling,Parchi et al. (1996) Science 274:2079-20821. These findings suggesteddifferent proteolytic cleavage sites due to the different conformationof PrP^(Sc) molecules in different strains of prions. Alternatively, theobserved differences could be explained by formation of differentcomplexes with other molecules, forming distinct cleavage sites inPrP^(Sc) in different strains [Marsh and Bessen (1994) Phil Trans R SocLond B 343:413-414]. Some researchers have proposed that different prionisolates may differ in the glycosylation patterns of prion protein[Collinge, Sidle et al. (1996) Nature 383:685-690; Hill, Zeidler et al.(1997) Lancet 349:99-100]. However, the reliability of bothglycosylation and peptide mapping patterns in diagnostics of multipleprion strains is currently still debated [Collings, Hill et al. (1997)Nature 386:564; Somerville, Chong et al. (1997) Nature 386:564].

A number of methods exist for the detection of a protein in a sample,and specifically for the detection of PrP^(Sc). Assays to detectPrP^(Sc) are described in U.S. Pat. Nos. 5,565,186 and 5,792,901 andU.S. patent application Ser. No. 08/935,363, incorporated herein byreference, which describe and disclose immunoassay methods fordetermining the presence of PrP^(Sc) in a sample. Quality assurance,quality control, and reagent documentation are all critical issues indetermining the presence of infectious prions in a sample. Variationbetween assays can be reduced by the use of a common standard for thecalibration of the different methods. The basis of a calibration systemis a primary standard sample that provides both high sensitivity andreproducibility of detection to effectively and consistently analyzedifferent samples. A standard is indispensable in assigning an accuratetarget value to reference materials in an assay method. Standards arealso useful in testing reagents used in assays for reliability andeffectiveness.

There is a method of providing standardized, cost-effective assays forreproducibly testing sample materials for the presence of a prionprotein. Accordingly, there is a need for standards for the calibrationof assays to detect prions and as controls in the assays, to ensure highsensitivity and to reduce problems of irreproducibility betweendifferent samples, and to test the quality of reagents used in theassays.

SUMMARY OF THE INVENTION

The invention provides prion protein standards for use as referencematerials in assays to detect prion proteins in a sample, e.g. determinethe presence of prions in a sample from a mammalian brain. The standardis preferably specific to prions which infect a single species and morepreferably may be specific to a single infectious strain. However, thestandardized preparation may include multiple strains and allow fordetection of multiple prion strains simultaneously.

In one embodiment, the invention features a standard produced from apreparation of brains from a plurality of transgenic host mammalsgenetically manipulated to allow infection by prions which normally onlyinfect a genetically diverse species, i.e. would generally only effectan animal with a significantly different PrP gene. The host animals areinoculated with prions from the genetically diverse species, the brainshomogenized, and the sample standardized. The preparations may bestandardized in accordance with a number of characteristics, e.g. bycontrolling level of infection, time from inoculation until diseasesymptoms are noted, genetic background, the concentration of prions andthe like. In addition, prions isolated from infected animals may be usedto ensure consistency of prion concentration in a standardizedbackground by spiking the prion preparation with prions may be (a)produced synthetically, (b) isolated from nansgenic animals, and/or (c)obtained from cadavers.

In another embodiment, the standard is comprised of isolated prionsintroduced to a homogenized preparation of brain. The isolated prionsmay be initially produced by transgenic host mammals. These transgenicanimals have their genome manipulated with respect to genetic materialrelated to a PrP gene such that the animals are susceptible to infectionwith a prion which generally only infects an animal which is geneticallydiverse from the host transgenic mammals used to produce the prionproteins. The transgenic animals are inoculated with prions of agenetically diverse species. After sufficient incubation time, prionsare isolated from the transgenic animals and the isolated prions areintroduced to a homogenized brain preparation. Preferably, the brainpreparation is of a species genetically similar and more preferablygenetically the same as the species susceptible to infection by theisolated prion proteins.

In yet another embodiment of the invention, a plurality of differentstandards are assembled to create a kit which is useful as a standardfor multiple prion strains. These samples have many uses, for example,to test for the specificity of an agent, e.g. an antibody, thatrecognizes PrP^(Sc). However, the standardized preparation is preferablyused in the creation of a positive control when using transgenic animalsand/or immunoassays to test samples for prions. Prion standardscontaining prions from a plurality of different species can be used totest cross-reactivity of the agent between species. The differentsamples can be dispersed within a single agglomerated sample, and thespecificity determined by the strength of the PrP^(Sc) recognition, orthe standards may be in a discrete assembly, allowing the elucidation ofreactivity to a standard of a specific species.

The invention also provides methods of preparing the prion proteinstandards. To produce the prion protein standard it is necessary toproduce a group of nonhuman host mammals which each have their genomemanipulated in an identical manner with respect to genetic materialrelated to a PrP gene such that the mammals are susceptible to infectionwith a prion which generally only infects an animal which is geneticallydiverse from the host. The transgenic host animals produced areinoculated with a prion containing composition that infects thegenetically diverse animal, and the animals are observed until theyexhibit symptoms of prion infection. Brain or other tissue is harvestedfrom the animals and homogenized to create the prion standard. Thisprocess is repeated, using homogenized brain tissue of a standardizedpreparation of a previously inoculated group to inoculate a new group,to further reduce variability in the production of the standard.Preferably, the inocula is from the group just prior to the new group.Different forms of transgenic animals can be used in the production ofdifferent preparations and two or more different standardizedpreparations can be mixed. However, it is preferable to produce thepreparation using genotypically similar non-human mammals withendogenous PrP gene ablated and having operatively inserted into itsgenome one or more of the following: an exogenous PrP gene from agenetically diverse species; an artificial PrP gene which includes aportion of the PrP gene of a genetically diverse species; and anartificial PrP gene with critical codons from a genetically diversespecies.

The invention also features a method of using a standard of theinvention as a positive control in a prion protein assay. The assay maybe a bioassay which uses transgenic animals (e.g. see U.S. Pat. No.5,792,901 issued Aug. 11, 1998) or an immunoassay (e.g. see PCTUC96/12510). The standards function to ensure reproducibility andspecificity of an assay by functioning as a reference material with aknown and consistent level of prion protein concentration. The standardsalso make it possible to determine sensitivity and to adjust selectivityrelative to sensitivity as needed.

The invention also features a method of calibrating an assay using thestandards of the invention. Calibration can be within a single assay, todetermine efficacy at a given level of prion protein concentration, orbetween assays, to allow comparison of results of different assays byadjusting detection levels between assays. For example, if one assay ismore sensitive than another, calibration with a standard can be used todetermine the factor for converting measured levels to corrected levelsfor comparison of results obtained using the different assays.

The invention also features a method of determining the quality ofreagents used in a prion protein assay by testing the reagents usingstandards of the invention. The standards provide a consistent prionprotein concentration and preferably a consistent background. Testingreagents against the standard can ensure selectivity and/orreproducibility of a reagent used in an assay.

The invention further provides a kit containing the standard andreagents needed to practice different types of bioassays andinmnunoassays. The reagents will vary depending on the assay, e.g. thereagents in an immunoassay may include the 3F4 antibody and/or the R1antibody as well as the standard. The kit may contain standards fordifferent prion strains and/or with different concentrations of prionsthat infect a single species of animal. Alternatively, the kit couldcontain standards for multiple species preferably with the same knownamount of prions in each standard, more preferably with each standardcontaining one or more infectious unit of prion proteins.

An object is to provide a standard generated from standardized prionpreparation produced from harvested brain tissue taken from animals thathave substantially identical genomes and specifically have substantiallyidentical genetic material related to prions, which animals exhibitsymptoms (in 250 days or less) of prion infection after being inoculatedwith prions which generally only infect a genetically diverse species.

A feature of the invention is that the standard itself may be used toinoculate new animals for the production of an additional prionstandard. This allows reproducibility of the standard between differentbatches, minimizes variability between different batches of thestandard, and allows for genetic homogeneity in the background used withthe standard.

An advantage of the invention is that the prion standard can eliminatethe need for extracting brain tissue from individual mammals for use aspositive controls in prion assays. Currently, control animals may havebeen infected with different types of prions and may each have adifferent genetic make up regarding genetic material related to prions.The use of a prion standard eliminates the variability of the control,both within a single experiment and between assays.

Another advantage is that prion assays can be carried out morereproducibly using assays calibrated by the prion standard of theinvention.

Another advantage is that the prion standard will allow comparison ofPrP^(Sc) levels determined by different prion assays.

Another advantage is that the prion standard will reduce variability ofprion assays, both within a single experiment and between multipleassays.

A feature of the present invention is that the plurality of transgenicand/or hybrid animals used to make the prion standard injected with asample containing pathogenic prions will consistently develop thedisease effects of the prions within a latively short time, e.g. about200 days±50 days after injection or less.

Another feature of the invention is that the transgene used in theproduction of the standard can be chosen to reflect any polymorphismsand/or mutations of the test material, resulting in a standard that isgenetically similar to the test material.

These and other objects, advantages and features of the invention willbecome apparent to those skilled in the art upon reading thisdisclosure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present standards and methods are disclosed and described, itis to be understood that this invention is not limited to particularantibodies, proteins, labels, assays or methods as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

The publications discussed herein are provided solely for the disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.Further, the dates of the publications provided are subject to change ifit is found that the actual date of publication is different from thatprovided here.

DEFINITIONS

The term “standard”, “prion protein standard”, “prion standard” and thelike as used herein refers to a preparation for prion assays in whichthe level of prion infection and background characteristics aresufficiently established to allow the standard to function as areference material, e.g., for prion immunoassays and/or prion bioassays.Such a standard is produced using the procedure of the invention, andthe resulting standard is reliable for both precision and accuracy inassays for determining prion concentration, infectivity, and the like.

The term reference material is used herein to describe a material havingone or more properties sufficiently well established to be used forcalibration of an apparatus or assay, for the assessment of ameasurement methodology, and for the verification of one or moremeasurements in other materials. Such properties may be prion proteinconcentration, infectious units of prions, genetic identity, backgroundprotein concentration, and the like. For example, a standardized prionpreparation with an established concentration of prion can be used as areference material for an assay to determine prion concentration in asample potentially infected with prions, or to determine that aparticular assay conforms to a desired detection level.

The term “sensitivity” as used herein refers to the relative strength ofrecognition of a protein by a binding partner, e.g. an antibody orantisense molecule. The recognition of the prion protein must besignificantly greater than the recognition of background proteins andpreferably the strength of recognition of the prion protein by thebinding partner is at least 10 times more preferably at least 100 timesgreater, and even more preferably at least 500 times greater thanrecognition of background proteins.

The term “selectivity” as used herein refers to the preferential bindingof a binding partner for an epitope on a prion protein. Preferably, aprion protein binding partner is at least 10 times, more preferably 100times, and even more preferably 1000 times more likely to bind to anepitope on a prion protein than to any epitope on a background protein.

The term infectious unit as used herein refers to an amount of prionswhich is capable of causing a prion-mediated infectious disease in ananimal upon exposure to this amount. Exposure to an amount of prionsbelow one infectious unit will generally not result in infectivity ofthose prions, and thus amounts below one infectious unit may not bedetected in a prion in vivo bioassay, i.e., only a hypersensitivebioassay will detect such. Generally, a single infectious unit isapproximately 10⁵ PrP molecules, although the number may vary with thevirulence of the prion strain.

The term “protein” as used herein is intended to encompass any aminoacid sequence and include modified sequences such as glycoproteins. Thetern includes naturally occurring proteins and peptides as well as thosewhich are recombinantly or synthetically synthesized. As used inconnection with the present invention the term “protein” is specificallyintended to cover naturally occurring proteins which occur in at leasttwo different conformations wherein both conformations have the same orsubstantially the same amino acid sequence but have different threedimensional structures. The two conformations of the protein include atleast one conformation which is not related to a disease state and atleast one conformation which is related to a disease state—pathogenic. Aspecific and preferred example of a protein as used in connection withthis disclosure is a PrP protein which includes the non-disease formreferred to as the PrP^(C) form and the disease related form referred asthe PrP^(Sc). Although a prion protein or the PrP^(Sc) form of a PrPprotein is infectious and pathogenic, the disease conformation of otherproteins is not infectious although it is pathogenic. As used herein,the term pathogenic may mean that the protein actually causes thedisease or it may simply mean that the protein is associated with thedisease and therefore is present when the disease is present. Thus, apathogenic protein as used in connection with this disclosure is notnecessarily a protein which is the specific causative agent of adisease.

The terms “PrP protein”, “PrP” and like are used interchangeably hereinand shall mean both the infectious particle form PrP^(Sc) known to causediseases (spongiform encephalopathies) in humans and animals and thenoninfectious form PrP^(C) which, under appropriate conditions isconverted to the infectious PrP^(Sc) form.

The terms prion, prion protein” and PrP^(Sc) protein” and the like areused interchangeably herein to refer to the infectious PrP^(Sc) form ofPrP, and is a contraction of the words “protein” and infection.”Particles are comprised largely, if not exclusively, of PrP^(Sc)molecules encoded by a PrP gene. Prions are distinct from bacteria,viruses and viroids. Known prions infect animals to cause scrapie, atransmissible, degenerative disease of the nervous system of sheep andgoats, as well as bovine spongiform encephalopathy (BSE), or “mad cowdisease”, and feline spongiform encephalopathy of cats. Four priondiseases known to affect humans are (1) kuru, (2) Creutzfeldt-JakobDisease (CJD), (3) Gerstinann-Sträussler-Scheinker Disease (GSS), and(4) fatal familial insomnia (FFI). As used herein prion” includes allforms of prions causing all or any of these diseases or others in anyanimals used—and in particular in humans and domesticated farm animals.

The term “PrP gene” is used herein to describe genetic material whichexpresses proteins including known polymorphisms and pathogenicmutations. The term “PrP gene” refers generally to any gene of anyspecies which encodes any form of a PrP protein. Some commonly known PrPsequences are described in Gabriel et al., Proc. Natl. Acad. Sci. USA89:9097-9101 (1992), and U.S. Pat. Nos. 5,565,186; 5,763,740; 5,792,901;and WO97/04814, incorporated herein by reference to disclose anddescribe such sequences. The PrP gene can be from any animal, includingthe “host” and “test” animals described herein and any and allpolymorphisms and mutations thereof, it being recognized that the termsinclude other such PrP genes that are yet to be discovered. The proteinexpressed by such a gene can assume either a PrP^(C) (non-disease) orPrP^(Sc) (disease) form.

The term “artificial PrP gene” is used herein to encompass the term“chimeric PrP gene” as well as other recombinantly constructed geneswhich when included in the genome of a host animal (e.g., a mouse) willrender the mammal susceptible to infection from prions which naturallyonly infect a genetically diverse test mammal, e.g., human, bovine orovine. In general, an artificial gene will include the codon sequence ofthe PrP gene of the mammal being genetically altered with one or more(but not all, and generally less than 40) codons of the natural sequencebeing replaced with a different codon—preferably a corresponding codonof a genetically diverse mammal (such as a human). The geneticallyaltered mammal being used to assay samples for prions which only infectthe genetically diverse mammal. Examples of artificial genes are mousePrP genes encoding the sequence as shown in FIGS. 3, 4 and 5 of U.S.Pat. No. 5,565,186 with one or more different replacement codonsselected from the codons shown in these Figures for humans, cows andsheep replacing mouse codons at the same position, with the proviso thatnot all the mouse codons are replaced with differing human, cow or sheepcodons. Artificial PrP genes of the invention can include not onlycodons of genetically diverse animals, but may include codons and codonsequences associated with genetic prion diseases such as CJD and codonsand sequences not associated with any native PrP gene but which, wheninserted into an animal, render the animal susceptible to infection withprions which would normally only infect a genetically diverse animal.

The terms “chimeric gene,” “chimeric PrP gene”, “chimeric prion proteingene” and the like are used interchangeably herein to mean anartificially constructed gene containing the codons of a host animalsuch as a mouse with one or more of the codons being replaced withcorresponding codons from a genetically diverse test animal such as ahuman, cow or sheep. In one specific example the chimeric gene iscomprised of the starting and terminating sequence (i.e., N- andC-terminal codons) of a PrP gene of a mammal of a host species (e.g. amouse) and also containing a nucleotide sequence of a correspondingportion of a PrP gene of a test mammal of a second species (e.g. ahuman). A chimeric gene will, when inserted into the genome of a mammalof the host species, render the mammal susceptible to infection withprions which normally infect only mammals of the second species. Thepreferred chimeric gene disclosed herein is MHu2M which contains thestarting and terminating sequence of a mouse PrP gene and a non-terminalsequence region which is replaced with a corresponding human sequencewhich differs from a mouse PrP gene in a manner such that the proteinexpressed thereby differs at nine residues.

The term “antibody” stands for an immunoglobulin protein which iscapable of binding an antigen. Antibody as used herein is meant toinclude the entire antibody as well as any antibody fragments (e.g.F(ab)′, Fab, Fv) capable of binding the epitope, antigen or antigenicfragment of interest Antibodies for assays of the invention may beimmunoreactive or immunospecific for and therefore specifically andselectively bind to a protein of interest e.g., an A4β amyloid proteinor a PrP protein. Antibodies which are immunoreactive and immunospecificfor both the native non-disease form and the treated disease form butnot for the untreated disease form (e.g., for both native PrP^(C) andtreated PrP^(Sc) but not native PrP^(Sc)) may be used because the sampleis treated to remove, i.e., hydrolyze PrP^(C). Antibodies for PrP arepreferably immunospecific—e.g., not substantially cross-reactive withrelated materials. Some specific antibodies which can be used inconnection with the invention are disclosed in published PCT applicationWO 97/10505 which is incorporated herein by reference to disclose anddescribe antibodies. This published PCT application corresponds to USSN08/713,939. Antibodies disclosed in the PCT application which bindPrP^(Sc) can be used to carry out the basic assay of the presentinvention when the sample has been treated with dispase sufficiently tohydrolyze all or substantially all of the PrP^(C) present in the sample.Another useful antibody for binding to PrP^(C) is the monoclonalantibody 263K 3F4 produced by the hybridoma cell line ATCC HB9222deposited on Oct. 8, 1986 in the American Type Culture Collection, 12301Paddawn Drive, Rockville, Md. 20852 and disclosed and described in U.S.Pat. No. 4,806,627 issued Feb. 21, 1989—incorporated by reference todisclose antibodies which selectively bind PrP^(C). The term “antibody”encompasses all types of antibodies, e.g. polyclonal, monoclonal, andthose produced by the phage display methodology. Particularly preferredantibodies of the invention are antibodies which have a relatively highdegree of affinity for both native PrP^(C) and treated PrP^(Sc) but arelatively low degree of or substantially no binding affinity forPrP^(Sc). More specifically, antibodies of the invention preferably havefour times or more, more preferably fifteen times or more, and stillmore preferably 30 times or Ad more binding affinity for both nativePrP^(C) and denatured PrP^(Sc) as compared with the binding affinity fornative PrP^(Sc).

“Purified antibody” refers to that which is sufficiently free of otherproteins, carbohydrates, and lipids with which it is naturallyassociated. Such an antibody “preferentially binds” to a denatureddisease conformation of a protein such as the denatured PrP^(Sc) protein(or an antigenic fragment thereof), and does not substantially recognizeor bind to other antigenically unrelated molecules. A purified antibodyof the invention is preferably immunoreactive with and immunospecificfor a specific species and more preferably immunospecific for nativePrP^(C) and for denatured forms of PrP^(C) and PrP^(Sc) or,alternatively, for native or untreated PrP^(Sc).

“Antigenic fragment” of a protein (e.g., a PrP protein) is meant aportion of such a protein which is capable of binding an antibody.

By “binds specifically” is meant high avidity and/or high affinitybinding of an antibody to a specific polypeptide e.g., epitope of aprotein, e.g., denatured PrP^(Sc) or denatured A4β protein. Antibodybinding to its epitope on this specific polypeptide is preferablystronger than binding of the same antibody to any other epitope,particularly those which may be present in molecules in associationwith, or in the same sample, as the specific polypeptide of intereste.g., binds more strongly to epitope fragments of a protein such asPrP^(Sc) so that by adjusting binding conditions the antibody bindsalmost exclusively to an epitope site or fragments of a desired proteinsuch as an epitope fragment exposed by denaturing of PrP^(Sc) and notexposed on native PrP^(Sc).

By “detectably labeled antibody”, “detectably labeled anti-PrP” or“detectably labeled anti-PrP fragment” is meant an antibody (or antibodyfragment which retains binding specificity), having an attacheddetectable label. The detectable label is normally attached by chemicalconjugation, but where the label is a polypeptide, it couldalternatively be attached by genetic engineering techniques. Methods forproduction of detectably labeled proteins are well known in the art.Detectable labels known in the art, but normally are radioisotopes,fluorophores, paramagnetic labels, enzymes (e.g., horseradishperoxidase), or other moieties or compounds which either emit adetectable signal (e.g., radioactivity, fluorescence, color) or emit adetectable signal after exposure of the label to its substrate. Variousdetectable label/substrate pairs (e.g., horseradishperoxidaseldiaminobenzidinc, avidin/streptavidin, luciferase/luciferin),methods for labeling antibodies, and methods for using labeledantibodies are well known in the art (see, for example, Harlow and Lane,eds. (Antibodies: A Laboratory Manual (1988) Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY)). Europium is a particularlypreferred label.

The terms “host animal” and “host mammal” are used to describe animalswhich will have their genome genetically and artificially manipulated soas to include genetic material which is not naturally present within theanimal. For example, host animals include mice, hamsters and rats whichhave their endogenous PrP gene altered by the insertion of an artificialgene of the present invention or by the insertion of a native PrP geneof a genetically diverse test animal.

The terms “test animal” and “test mammal” are used to describe theanimal which is genetically diverse from the host animal in terms ofdifferences between the PrP gene of the host animal and the PrP gene ofthe test animal. The test animal may be any animal for which one wishesto run an assay test to determine whether a given sample contains prionswith which the test animal would generally be susceptible to infection.For example, the test animal may be a human, cow, sheep, pig, horse,cat, dog or chicken, and one may wish to determine whether a particularsample includes prions which would normally-only infect the test animal.This is done by including PrP gene sequences of the test animal into thehost animal and inoculating the host animal with prions which wouldnormally only infect the test animal.

The terms “genetically diverse animal” and “genetically diverse mammal”are used to describe an animal which includes a native PrP codonsequence of the host animal which differs from the genetically diversetest animal by 17 or more codons, preferably 20 or more codons, and mostpreferably 28-40 codons. Thus, a mouse PrP gene is genetically diversewith respect to the PrP gene of a human, cow or sheep, but is notgenetically diverse with respect to the PrP gene of a hamster.

The terms “ablated PrP gene”, “disrupted PrP gene”, “ablated PrP gene”and the like are used interchangeably herein to mean an endogenous PrPgene which has been altered (e.g., add and/or remove nucleotides) in amanner so as to render the gene inoperative. Examples of non-functionalPrP genes and methods of making such are disclosed in Büeler, H., et al“Normal development of mice lacking the neuronal cell-surface PrPprotein” Nature 356:577-582 (1992) which is incorporated herein byreference. Both alleles of the genes are preferably disrupted.

The terms “hybrid animal”, “transgenic hybrid animal” and the like areused interchangeably herein to mean an animal obtained from thecross-breeding of a first animal having an ablated endogenous PrP genewith a second animal which includes either (1) a chimeric gene orartificial PrP gene or (2) a PrP gene from a genetically diverse animal.For example a hybrid mouse is obtained by cross-breeding a mouse with anablated mouse PrP gene with a mouse containing (1) bovine PrP genes(which may be present in high copy numbers) alone or with (2) chimericPrP genes. The term hybrid includes any offspring of a hybrid includinginbred offspring of two hybrids provided the resulting offspring issusceptible to infection with prions with normal infect only agenetically diverse species and the symptoms of the infection areobservable in about 350 days or less, preferably 250 or less.

The terms “susceptible to infection” and “susceptible to infection byprions” and the like are used interchangeably herein to describe atransgenic or hybrid test animal of the invention which develops a priondisease if inoculated with options which would normally only infect agenetically diverse test animal. The terms are used to describe atransgenic or hybrid animal of the invention such as a transgenic mouseTg(MHu2M) which, without the chimeric PrP gene, would not be susceptibleto infection with a human prion (less than 20/chance of infection) butwith the chimeric gene is susceptible to infection with human prions(80% to 100% chance of infection). If an animal is susceptible toinfection with a particular prion that animal, if inoculated with theprion, will show symptoms of prion disease infection in about 350,preferably 250 days or less.

The teen “incubation time” shall mean the time from inoculation of ananimal with a prion until the time when the animal first developsdetectable symptoms of disease resulting from the infection. A reducedincubation time is one year or less, preferably about 200 days±50 daysor less, more preferably about 50 days±20 days or less.

The terms “standardized prion preparation”, “prion preparation”,“preparation” and the like are used interchangeably herein to describe acomposition containing prions which composition is obtained from braintissue of mammnals which contain substantially the same genetic materialas relates to prions, e.g., brain tissue from a set of mammals whichexhibit signs of prion disease which mammals (1) include a transgene ofthe invention; (2) have an ablated endogenous prion protein gene; (3)have a prion protein gene from a genetically diverse species; or (4) arehybrids with an ablated endogenous prion protein gene and a prionprotein gene from a genetically diverse species. The mammals from whichstandardized prion preparations are obtained exhibit clinical signs ofCNS dysfunction as a result of inoculation with prions and/or due todeveloping the disease due to their genetically modified make up, e.g.,species specific prion protein genes.

The term endogenous prion protein concentrations and “endogenous prionconcentration” as used herein refers to the concentration of theendogenous prion protein in the brain homogenate of the standard. Forexample, the endogenous prion protein of a standard generated usingprion ablated host animals should preferably be undetectable. In anotherexample, the endogenous prion protein of a standard generated usingbrain homogenate of a normal genetically diverse animal should only havePrP^(C) present, and these levels should be within the physiologicallynormal range of that animal, e.g. a standard using normal bovine brainshould have an endogenous prion concentration within the range of anormal bovine brain.

The term “exogenous prion protein concentration” as used herein refersto the concentration of prions, e.g prions of a genetically diversespecies, produced in a host mammal. The term refers to both PrP^(C) andPrP^(Sc), or a combination of the two depending on the context. Theexogenous prion protein concentration refers to the level of exogenousprions in the brain homogenate of host animals infected with prions thatnormally infect a genetically diverse species. Alternatively, exogenousprion protein concentration can refer to the concentration of isolatedprion protein introduced into the standard.

The term “background protein concentration” as used herein refers tolevels of any background protein, i.e. any protein other than prionspresent in the brain preparation used to generate the standard.

The term “assay value” as used herein refers to the levels of priondetected in a sample, a standard, or a portion of a standard using anassay designed to detect prion concentration or infectivity.

The term “true value” as used herein refers to the level of prionprotein present in a sample that is detectable using reliable techniquesknown in the art for determining protein levels.

The term correction value as used herein refers to the numericaladjustment needed to convert the prion assay value to the true value.The correction value may be additive, multiplicative, or exponential,depending on the assay.

Abbreviations used herein include:

-   CNS for central nervous system;-   BSE for bovine spongiform encephalopathy;-   CJD for Creutzfeldt-Jacob Disease;-   FFI for fatal familial insomnia;-   GdnHCI for Guanidine hydrochloride;-   GSS for Gerstamnn-Strassler-Scheinker Disease;-   Hu for human;-   HuPrP for human prion protein;-   Mo for mouse;-   MoPrP for mouse prion protein;-   SHa for a Syrian hamster,-   SHaPrP for a Syrian hamster prion protein;-   Tg for transgenic;-   Tg(SHaPrP) for a tansgenic mouse containing a PrP gene of a Syrian    hamster,-   Tg(HuPrP) for transgenic mice containing the complete human PrP    gene;-   Tg(ShePrP) for transgenic mice containing the complete sheep PrP    gene;-   Tg(BovPrP) for transgenic mice containing the complete cow PrP gene;-   PrP^(Sc) for the scrapie isoform of the prion protein;-   PrP^(C) for the cellular contained common, normal isoform of the    prion protein;-   PrP 27-30 or PrP^(Sc) 27-30 for the treatment or protease resistant    form of PrP^(Sc);-   MoPrP^(Sc) for the scrapie isoform of the mouse prion protein;-   MHu2M for a chimeric mouse/human PrP gene wherein a region of the    mouse PrP gene is replaced by a corresponding human sequence which    differs from mouse PrP at 9 codons;-   Tg(MHu2M) mice are transgenic mice of the invention which include    the chimeric MHu2M gene;-   MHu2MPrP^(Sc) for the scrapie isoform of the chimeric human/mouse    PrP gene;-   PrP^(CJD) for the CJD isoform of a PrP protein;-   Prnp^(0/0) for ablation of both alleles of an endogenous prion    protein gene, e.g., the MoPrP gene;-   Tg(SHaPrP^(+/0))81/Prnp^(0/0) for a particular line (81) of    transgenic mice expressing SHaPrP, +/0 indicates heterozygous;-   Tg(HuPrP)/Prnp^(0/0) for a hybrid mouse obtained by crossing a mouse    with a human prion protein gene (HuPrP with a mouse with both    alleles of the endogenous prion protein gene disrupted;-   Tg(MHu2M)/Prnp^(0/0) for a hybrid mouse obtained by crossing a mouse    with a chimeric prion protein gene (MHu2M) with a mouse with both    alleles of the endogenous prion protein gene disrupted;-   FVB for a standard inbred strain of mice often used in the    production of transgenic mice since eggs of FVB mice are relatively    large and tolerate microinjection of exogenous DNA relatively well.

STANDARDIZED PREPARATIONS—IN GENERAL

Standardized preparations are generally characterized by containing aknown amount of a disease related conformation of a protein. The proteinmay be any protein with two or more three dimensional conformations andis preferably a composition containing a known amount of a PrP proteinin its disease conformation, i.e. a known amount of PrP^(Sc). The amountof PrP^(Sc) may be an amount in terms of infectious units of PrP^(Sc),concentration of PrP^(Sc), or number of molecules of PrP^(Sc)present ina unit volume of the sample. An array of preparations containingdifferent amounts of PrP^(Sc) and/or different strains would provide auseful kit for bioassays or immunoassays.

A standardized prion preparation of the invention is comprised of: (1)prions obtained from a plurality of different sources, e.g., a pluralityof genetically identical tnansgenic mice and (2) a carrier which is notthe brain tissue of the animals normally infected by the prions. Theprions are of a known strain, present in a known amount and infect andcause disease in a known species of animal. The prions are preferablyobtained from the brains of 10 or more transgenic mice which have beengenetically manipulated so that they are injectable with a specificstrain of prions which generally only infects a human, cow or sheep.

Standardization of assays to detect prion proteins requires ademonstration of precision and accuracy in the measurement of prionprotein in a sample. Precision requires that prion concentrationsobtained in replicate assays should be in good agreement within aselected standard of error. Preferably, the standard of error is10^(0.2) at ID₅₀ units/ml, where ID50 unit is defined as the infectiousdose at which 50% of the test animals develop prion disease. Precisioncan be obtained by quality and consistency of reagents and protocolsused in the assays. Accuracy requires that the concentration obtained inthe assay should either reflect the true concentration of the prionprotein in the sample, or that the true concentration can bereproducibly determined by altering the obtained value by a constantfactor. Accuracy is best optimized by careful and consistentmethodology, quality of technical determination of proteinconcentrations, and a minimization of error.

In addition, if different methods are used to detect prion protein,standardization requires a harmonization of the data obtained using thedifferent methods. Different protocols to determine prion proteinconcentrations may vary with respect to a number of factors, for examplethe storage of the sample, the preparation of the sample prior tovisualization of the protein, the chemicals used in the processing ofthe sample, and the like. Many potential changes in prion protein levelsfrom obtaining, storing or preparing samples for prion assays aremethod-dependent. Harmonization of data can be achieved by usingsuitable standard reference materials. To be suitable for harmonization,reference standards should have the same immunochemical behavior as thesamples to be analyzed in all methods. In addition, it is crucial thatthe standards be consistent, i.e. the prion concentration does notnoticeably vary in different samples of the standard, and reproducible,i.e. the values obtained using different samples of the standard do notvary outside a standard of error. The reference standard may have anumber of different physical forms, and may be lyophilized,liquid-stabilized, frozen, etc.

Standardized Prion Preparation

Prion standards are produced for use in assays so as to determine thespecificity, sensitivity and/or reliability of the assay. Standards areproduced using standardized prion preparations from any host animal,although preferably the preparations are obtained from a host animalwhich has brain material containing prions of a test animal. Forexample, a Tg mouse containing a human prion protein gene can producehuman prions and the brain of such a mouse can be used to create astandardized human prion preparation. Further, in that the preparationis to be a “standard” it is preferably obtained from a battery (e.g.,100, 500, 1,000, or more animals) of substantially identical animals.For example, 100 mice all containing a very high copy number of humanPrP genes (all polymorphisms and mutations) would spontaneously developdisease and the brain tissue from each could be combined to make auseful standardized human prion preparation. The preparation ispotentially infinite in size because substantially identicalpreparations can be produced at any time by following an establishedprotocol.

Standardized prion preparations can be produced using any of themodified host mammals of the present invention. For example,standardized prion preparations could be produced using mice, rats,hamsters, or guinea pigs which are genetically modified per the presentinvention so that they are susceptible to infection with prions whichprions would generally only infect genetically diverse species such as ahuman, cow, sheep or horse and which modified host mammals will developclinical signs of CNS dysfunction within a period of time of 350 days orless after inoculation with prions. The most preferred host mammal is amouse in part because they are inexpensive to use and because a greateramount of experience has been obtained with respect to production oftransgenic mice than with respect to the production of other types ofhost animals.

Once an appropriate type of host is chosen, such as a mouse, the nextstep is to choose the appropriate type of genetic manipulation to beutilized to produce a standardized prion formulation. For example, themice may be mice which are genetically modified by the insertion of achimeric gene of the invention. Within this group the mice might bemodified by including high copy numbers of the chimeric gene and/or bythe inclusion of multiple promoters in order to increase the level ofexpression of the chimeric gene. Alternatively, hybrid mice of theinvention could be used wherein mice which have the endogenous PrP geneablated are crossed with mice which have a human PrP gene inserted intotheir genome. There are, of course, various subcategories of such hybridmice. For example, the human PrP gene may be inserted in a high copynumber and/or used with multiple promoters to enhance expression. In yetanother alternative the mice could be produced by inserting multipledifferent PrP genes into the genome so as to create mice which aresusceptible to infection with a variety of different prions, i.e., whichgenerally infect two or more types of test animals. For example, a mousecould be created which included a chimeric gene including part of thesequence of a human, a separate chimeric gene which included part of thesequence of a cow and still another chimeric gene which included part ofthe sequence of a sheep. If all three different types of chimeric geneswere inserted into the genome of the mouse the mouse would besusceptible to infection with prions which generally only infect ahuman, cow and sheep.

After choosing the appropriate mammal (e.g., a mouse) and theappropriate mode of genetic modification (e.g., inserting a chimeric PrPgene) the next step is to produce a large number of such mammals whichare substantially identical in terms of genetic material related toprions. More specifically, each of the mice produced will include anidentical chimeric gene present in the genome in substantially the samecopy number. The mice should be sufficiently identical genetically interms of genetic material related to prions that 95% or more of the micewill develop clinical signs of CNS dysfunction within 350 days or lessafter inoculation and all of the mice will develop such CNS dysfunctionat approximately the same time e.g., within ±30 days of each other.

Once a large group e.g., 50 or more, more preferably 100 or more, stillmore preferably 500 or more of such mice are produced. The next step isto inoculate the mice with prions which generally only infect agenetically diverse mammal e.g., prions from a human, sheep, cow orhorse. The amounts given to different groups of mammals could be varied.After inoculating the mammals with the prions the mammals are observeduntil the mammals exhibit symptoms of prion infection, e.g., clinicalsigns of CNS dysfunction. After exhibiting the symptoms of prioninfection the brain or at least a portion of the brain tissue of each ofthe mammals is extracted.

The extracted brain tissue is homogenized which provides thestandardized prion preparation.

As an alternative to inoculating the group of transgenic mice withprions from a genetically diverse animal it is possible to produce micewhich spontaneously develop prion related diseases. This can be done,for example, by including extremely high copy numbers of a human PrPgene into a mouse genome. When the copy number is raised to, forexample, 100 or more copies, the mouse will spontaneously developclinical signs of CNS dysfunction and have, within its brain tissue,prions which are capable of infecting humans. The brains of theseanimals or portions of the brain tissue of these animals can beextracted and homogenized to produce a standardized prion preparation.

The standardized prion preparations of the invention can be useddirectly or can be diluted and titered in a manner so as to provide fora variety of different positive controls. More specifically, variousknown amounts of such standardized preparation can be used to inoculatea first set of transgenic control mice. A second set of substantiallyidentical mice are inoculated with a material to be tested, i.e., amaterial which may contain prions. A third group of substantiallyidentical mice are not injected with any material. The three groups arethen observed. The third group, should, of course, not become ill inthat the mice are not injected with any material. If such mice do becomeill the assay is not accurate probably due to the result of producingmice which spontaneously develop disease. If the first group, injectedwith a standardized preparation, do not become ill the assay is alsoinaccurate probably because the mice have not been correctly created soas to become ill when inoculated with prions which generally only infecta genetically diverse mammal. However, if the first group does becomeill and the third group does not become ill the assay can be presumed tobe accurate. Thus, if the second group does not become ill the testmaterial does not contain prions and if the second group does become illthe test material does contain prions.

By using standardized prion preparations of the invention it is possibleto create extremely dilute compositions containing the prions. Forexample, a composition containing one part per million or less or evenone part per billion or less can be created. Such a composition can beused to test the sensitivity of the transgenic mice of the invention indetecting the presence of prions in the sample.

Prion preparations of the present invention are desirable in that theywill include a constant amount of prions and are extracted from anisogeneic background. Accordingly, contaminates in the preparations willbe constant and controllable. Standardized prion preparations of theinvention will be useful in the carrying out of bioassays in order todetermine the presence, if any, of prions in various pharmaceuticals,whole blood, blood fractions, foods, cosmetics, organs and in particularany material which is derived from an animal (living or dead) such asorgans, blood and products thereof derived from living or dead humans.Thus, standardized prion preparations of the invention will be valuablein validating purification protocols where preparations are spiked andreductions in teeter measured for a particular process.

TRANSGENIC ANIMALS AS PRION SOURCE

Prion protein standards of the invention are useful as standards ofinfectivity in an in vivo assay for prion infection. The host animalsused in these assays are genetically altered to be susceptible to prionswhich normally only infect a species genetically diverse from the hostanimal. The DNA sequence of the human, sheep and cow PrP genes have beendetermined allowing, in each case, the prediction of the complete aminoacid sequence of their respective prion proteins. The normal amino acidsequence which occurs in the vast majority of individuals is referred toas the wild-type PrP sequence. This wild-type sequence is subject tocertain characteristic polymorphic variations. In the case of human PrP,two polymorphic amino acids occur at residues 129 (Met/Val) and 219(Glu/Lys). Sheep PrP has two amino acid polymorphisms at residues 171and 136, while bovine PrP has either five or six repeats of an eightamino acid motif sequence in the amino terminal region of the matureprion protein. While none of these polymorphisms are of themselvespathogenic, they appear to influence prion diseases. Distinct from thesenormal variations of the wild-type prion proteins, certain mutations ofthe human PrP gene which alter either specific amino acid residues ofPrP or the number of octarepeats have been identified which segregatewith inherited human prion diseases. These polymorphisms and mutationsare summarized in the following table:

MUTATION TABLE Pathogenic human Human Sheep Bovine mutationsPolymorphisms Polymorphisms Polymorphisms 2 octarepeat insert Codon 129Codon 171 5 or 6 octarepeats Met/Val Arg/Glu 4 octarepeat insert Codon219 Codon 136 Glu/Lys Ala/Val 5 octarepeat insert 6 octarepeat insert 7octarepeat insert 8 octarepeat insert 9 octarepeat insert Codon 102Pro-Leu Codon 105 Pro-Leu Codon 117 Ala-Val Codon 145 Stop Codon 178Asp-Asn Codon 180 Val-Ile Codon 198 Phe-Ser Codon 200 Glu-Lys Codon 210Val-Ile Codon 217 Asn-Arg Codon 232 Met-Ala

Mutations and polymorphisms in the genes of genetically diverse animalscan be introduced to host animals to create the standards of theinvention. For example, a chicken, bovine, sheep, rat and mouse PrP geneare disclosed and published within Gabriel et al., Proc. Natl. Acad.Sci. USA 89:9097-9101 (1992). The sequence for the Syrian hamster ispublished in Basler et al., Cell 46:417-428 (1986). The PrP gene ofsheep is published by Goldmann et al., Proc. Natl. Acad. Sci. USA87:2476-2480 (1990). The PrP gene sequence for bovine is published inGoldmann et al., J. Gen. Virol. 72:201-204 (1991). The sequence forchicken PrP gene is published in Harris et al., Proc. Natl. Acad. Sci.USA 88:7664-7668 (1991). The PrP gene sequence for mink is published inKretzschmar et al., J. Gen. Virol. 73:2757-2761 (1992). The human PrPgene sequence is published in Kretzschmar et al., DNA 5:315-324 (1986).The PrP gene sequence for mouse is published in Locht et al., Proc.Natl. Acad. Sci. USA 83:6372-6376 (1986). The PrP gene sequence forsheep is published in Westaway et al., Genes Dev. 8:959-969 (1994).Further PrP sequences and differences between sequences and knownmutations are disclosed in U.S. Pat. No. 5,792,901 issued Aug. 11, 1998.These publications are all incorporated herein by reference to discloseand describe the PrP gene and PrP amino acid sequences that may be usedin the generation of the standards of the invention.

In one preferred embodiment of the invention, the test animal used inthe assay is Tg(HuPrP)Prnp^(0/0), and the prion protein standardproduced for this assay is generated using this strain of mouse. TheHuPrP construct may vary with respect to known polymorphisms as well asknown pathogenic mutations. Thus, when the genetic material isexpressed, the resulting protein will be HuPrP. After the human PrPtransgene is produced, it can be microinjected into a mouse egg usingknown technology as described within Scott et al., Cell 59:847-857(1989) and Scott et al., Protein Sci. 1:986-997 (1992) and see alsoWO91/19810 published Dec. 22, 1991 as well as other publicationsrelating to the production of transgenic mice cited therein and known tothose skilled in the art.

In another preferred embodiment, the test animal is a mouse with anablated endogenous PrP gene and an exogenous bovine PrP gene,Tg(BovPrP)/Prnp^(0/0). A construct containing the full-length bovine PrPgene is stably introduced to the genome of a PrP^(0/0) mouse bymicroinjection of the construct into a PrP^(0/0) egg. The injected mouseegg is then implanted into a mouse using known procedures. Multiple eggscan be implanted into a single mouse and known procedures can be used todetermine whether the resulting offspring are transgenic mice whichinclude the transgene within their genome.

Quality Control of Prion Protein Standards

Once the standard of the invention is prepared, it nee&s to undergo aseries of tests and controls to check the established properties of thestandard sufficiently well established to allow use of the standard. Theproperties should be determined not only for the new batch, but also forconsistency between different aliquots of the batch. For a prion proteinstandard, properties such as prion concentration, antigenicity,background elements, and the like.

Moreover, the standard needs to be stored in such a manner that itpreserves its initial chemical, physical and biological properties overtime. Thus, the standard should be stored in a manner that minimizesbiodegradation, chemical transformations, change of the oxidative stateof portions of the sample, interaction with the storage container, andother reactions that may take place during storage. Tests to evaluatepossible changes occurring during storage can be performed by analyzingthe materials at different times.

The levels of prion in a sample can also be maintained by “spiking” thestandard with the appropriate isolated prion protein, i.e. addingpurified prion protein in order to maintain a desired level of prionprotein in a sample. The added protein may be either PrP^(C) or PrP^(Sc)as necessary to achieve the desired parameters of the standard, and itmay be either the whole protein or the segment that is infective (e.g.for the in vivo infectivity assay) or the fragment that is antigenic(e.g. for an immunoassay). These spiked materials may be very useful inenhancing laboratory performance, especially for analytical methods forspecific types of materials such as prions, which may be at extremelylow concentrations in the initial stages of infection.

Spiked in Vivo Material as Prion Standard

In another embodiment, normal or diseased tissue from a mammal is spikedwith prions that normally infect that species. The isolated prions areproduced from transgenic mice that are susceptible to infection byprions from the genetically diverse species. For instance, normal bovinebrain can be spiked with prions harvested from Tg(BovPrP)PrP^(0/0) mousewhich correspond to prions that naturally infect cows. In anotherexample, the brain homogenate is prepared from the brain of a cowsuffering from BSE. The initial inocula used to infect theTg(BovPrP)PrP^(0/0) preferably comes from a cow genetically similar tothe cow brain being used as the preparation material for the standard.This embodiment of the invention may be preferable for an assay in whichthe background (e.g. proteins, etc.) of cow brain is extremelyimportant, but a standardized concentration of prion and/or infectivitylevel is needed in order to standardize a procedure, test a reagent, andthe like.

Standards to Test Efficacy of Reagents for Assays

The standards of the invention provide a practical means for uniformtesting of antibodies and other reagents used in immunoassays underdefined conditions. A variety of different types of assays of theinvention may be used with one or more different agents that recognizeprions, such as antibodies. These antibodies can be tested forimmunoreactivity, specificity for a particular conformation, and/orcross-reactivity with prions from different species using the standardsof the invention. For example, each new antibody, whether it is a newmonoclonal antibody to a different epitope, a new hybridoma producing anantibody predicted to recognize a known epitope, or a new batch ofpolyclonal antibodies, can be evaluated for immunoreactivity using aprion standard preparation. The availability of a standard preparationof prion proteins would permit producers of reagents used in the assaysto establish an internal quality control program. In addition, havingthe standard used to determine the specificity and reproducibility ofthe reagents available to those performing the assay would allowlaboratories to conduct performance testing of all assays usingstandardized reagents.

Methods of generating antibodies are generally known to those skilled inthe art. In that the disease form is often in a tighter configurationthan the non-disease form, with less epitopes exposed, one can readilygenerate antibodies which bind only to the non-disease form of theprotein or the treated disease form. For example, antibodies detectingtreated forms of PrP^(Sc) protein and PrP^(C) protein may be generatedby immunizing rabbits or mice with a-helical conformations ofrecombinant PrP, native PrP^(C) from animal brains, synthetic peptidesin a-helical or random coil conformations, or against denatured PrP^(Sc)or PrP 27-30. Only antibodies with affinity at least 4 fold higher forPrP^(C) (or denatured conformation of PrP^(Sc)of the same species) ascompared to their affinity for PrP^(Sc) should be selected. The methodof antibody generation, purification, labeling and detection may vary.An antibody which can be used in the invention is disclosed in U.S. Pat.No. 4,806,627, issued Feb. 21, 1989, disclosing monoclonal antibody 263K3F4, produced by cell line ATCC HB9222 deposited on Oct. 8, 1986, whichis incorporated herein by reference. The cell line producing theantibody can be obtained from the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852.

In general, scrapie infection fails to produce an immune response, withhost organisms being tolerant to PrP^(Sc) from the same species.Antibodies which bind to either PrP^(C) or PrP^(Sc) are disclosed inWO97/10505, published Mar. 20, 1997. Any antibody binding to PrP^(C) andnot to PrP^(Sc) can be used, and those skilled in the art can generatesuch using known procedures, e.g., see methods of producing page displayantibody libraries in U.S. Pat. No. 5,223,409. Polyclonal anti-PrPantibodies have though been raised in rabbits following immunizationwith large amounts of formic acid or SDS-denatured SHaPrP 27-30[Bendheim, Barry et al. (1984) Nature 310:418-421; Bode, Pocchiari etal. (1985) J Gen Virol 66:2471-2478; Safar, Ceroni et al. (1990)Neurology 40:513-517]. Similarly, a handful of anti-PrP monoclonalantibodies against PrP 27-30 have been produced in mice [Barry andPrusiner (1986) J Infect Dis 154:518-521; Kascsak, Rubenstein et al.(1987) J Virol 61:3688-3693]. These antibodies were generated againstformic acid- or SDS-denatured PrP 27-30 and are able to recognize nativePrP^(C) and treated or denatured PrP^(Sc) from both SHa and humansequally well, but do not bind to MoPrP. Not surprisingly, the epitopesof these antibodies were mapped to regions of the sequence containingamino acid differences between SHa- and MoPrP [Rogers, Yehiely et al.(1993) Proc Natl Acad Sci USA 90:3182-3186].

Antibodies secreted from a single hybridoma can be tested to ensure thatthey retain the desired level of detection, since the standards of theinvention provide a sample with reproducible antigenicity. Accordingly,a hybridoma can be monitored for stability of production and, ifnecessary, a new hybridoma expressing the antibody of choice can beisolated and tested for a specific level of binding to the PrP geneproduct. Testing the antibodies against a standard and a control (e.g.the transgenic animals used to produce the standard that have not beeninfected) will also allow standardization of the cross-reactivity of theantibodies, which is especially useful in ensuring that antibodies suchas R1 bind to both conformations of the prion protein, or that anantibody such as 3F4 binds preferentially to one native conformation,but binds well to both conformations upon denaturation.

Multiprion Standards

In one embodiment of the invention, a standard may be used that providesa reference material for multiple strains of prion at one time. Thedifferent prions in the standard may be variants from a single species,or may contain prions from multiple species. The different prions in thestandard may be interspersed in the sample such that cross-reactivityand/or specificity is determined primarily through the intensity of thesignal produced, or by a double labeling procedure. For instance, if amultiprion standard contains different samples of prions that willinfect sheep, cows, and goats, with such prions being in roughly equalconcentration, an antibody that recognizes a conserved epitope on allthree prions will result in a signal three times as strong as anantibody that only recognizes an epitope specific to a sheep prion.Alternatively, the cross-reactivity of an antibody with multiple speciescan be determined by also subjecting the tissue to an antibody that isspecific to one of the prions in the multiprion standard.

In a preferred embodiment, a multiprion standard is provided in whichmultiple samples of prion of different variants and/or species arediscretely distributed in a single standard, allowing the identificationof the particular sample that reacts with an agent. One example of sucha standard is a checkerboard tissue block, which can serve as amultipurpose control for slides, the evaluation of new reagents,specificity of assays, etc. The physical structure of such a standard isdescribed in Rattifora and Matha, Lab Invest. 68:722-24 (1990) and inPetrosyan and Press, Lab Invest, 77:541-542 (1997), both of which areincorporated herein by reference.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use standards of the present invention, and are not intended tolimit the scope of what the inventors regard as their invention, nor arethey intended to represent or imply that the experiments below are allof or the only experiments performed. Efforts have been made to ensureaccuracy with respect to numbers used (e.g. amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreescentigrade, and pressure is at or near atmospheric.

Example 1 Creation of a Tg(BovPrP) Strain for Standard Preparation

When using a standard to assess the presence and infectivity of prionprotein within a bovine sample, it is desirable to have a standardhaving a known relatively constant amount of bovine prions. It is alsodesirable to have a standard with a PrP gene as genetically similar tothe bovine test material as possible. To this end, the prion standardfor testing bovine material for prion proteins can be produced using atransgenic mouse with its endogenous PrP gene replaced by an exogenousbovine PrP gene.

Transgenic mice having an endogenous Tg(BovPrP)/PrP^(0/0) mice arecreated and propagated as disclosed in U.S. Pat. No. 5,792,901, which isincorporated herein by reference. The bovine PrP gene is introduced toproduce Tg(BovPrP)/PrP^(0/0) that are susceptible to bovine prions fromcattle with BSE. The Tg(BovPrP)/PrP^(0/0) mice produce prions capable ofinfecting cows within their brain tissue. The brains of these mice orportions of the brain tissue of these animals are extracted andhomogenized to produce a standardized prion preparation for use as aprion protein standard. A 10% [w/v] homogenate of infected brain tissuefrom an infected Tg(BovPrP)/PrP^(0/0) mouse is prepared in phosphatebuffered saline lacking calcium and magnesium ions. The tissue isinitially dissociated using a sterile disposable homogenizer, and thissuspension was subjected to repeated extrusion through an 18 gaugesyringe needle followed by a 22 gauge needle. Samples to be used asstandards are assayed for desired properties, e.g. PrP^(Sc)concentration and overall prion concentration, are diluted 10-fold, andstored.

Example 2 Creation of a Tg(HuPrP) Strain for Standard Preparation

Transgenic mice having an endogenous PrP gene and an exogenouslyintroduced human PrP gene can be used to produce a standard fordetecting prions in human material. The human transgene Tg(HuPrP)introduced to the transgenic mice can encode any human prion strain,including known non-pathogenic polymorphisms, germline PrP mutations,known sporadic PrP mutations, etc. In this manner the PrP gene of thestandard may be designed to be genetically similar to the human testmaterial. Thus, the use of the term Tg(HuPrP) herein includes humantransgenes having different polymorphisms and/or mutations.

Human inocula are derived from frozen brain tissues of patients in whichthe clinical diagnosis of CJD, GSS, or FFI had been confirmed byhistopathological examination of brain tissues and, in most cases, byprion protein analysis. In some cases, the PrP gene was amplified by PCRof DNA isolated from patient blood and the PrP sequence determined byDNA sequence analysis. Human brain specimens are collected from patientsdying of sporadic, inherited or infectious prion disease. A 10% [w/v]homogenate of brain tissue from a patient diagnosed with CJD, GSS, orFFI is prepared in phosphate buffered saline lacking calcium andmagnesium ions. The tissue is initially dissociated using a steriledisposable homogenizer, and this suspension is subjected to repeatedextrusion through an 18 gauge syringe needle followed by a 22 gaugeneedle. Samples for inoculation into test animals are diluted 10-fold.

Tg(HuPrP/PrP^(0/0) mice are created and propagated as disclosed in U.SPat. No. 5,792,901, which is incorporated herein by reference. Thesemice are inoculated intracerebrally with 30 μl of infected brain extractusing a 27 gauge needle inserted into the right parietal lobe. Thepreparation of inocula and criteria for diagnosis of scrapie are asdescribed above in Example 1. Homogenate of either the brain of a singleinfected animal or a plurality of animals infected with the same inoculais then used to inoculate a larger number of Tg(HuPrP)/PrP^(0/0) mice,which are then followed for signs of infectivity. Depending on the levelof prion protein desired in the standard, animals can be killed at aspecific time following innoculation and/or when they exhibit a specificphysiological response to infection, e.g. a certain degree of ataxia.These brain samples are pooled, and a new batch of Tg(HuPrP)/PrP^(0/0)mice inoculated with the homogenate. This continues, with a new batch ofmice used for the production of the standard being inoculated withinocula from a preceding generation, and most preferably from theinfected mice directly preceding the new generation. Alternatively,several generations of mice can be infected with the inocula of a singleearlier generation. This procedure allows the standardization of theprion concentration of the standard while diminishing the background dueto genetic variation of the prion preparation.

Total protein concentrations in brain homogenates are determined bybicinchoninic acid assay. Immuno dot blots for the determination of therelative levels of PrP expression in Tg mouse brains are performed aspreviously described (Scott et al., 1993). Samples for Western blotanalyses are prepared by digesting brain homogenates with 20 μgproteinase K for 60 min at 37° C. Western blots are performed asdescribed previously in Barry, R. A., et al., “Monoclonal antibodies tothe cellular and scrapie prion proteins,” J. Infect. Dis., 154:518-521(1986); Towbin, H., et al., “Electrophoretic transfer of proteins frompolyacrylamide gels to nitrocellulose sheets: Procedure and someapplications,” Proc. Natl. Acad. Sci. USA 76:4350-4354 (1979), exceptthat an enhanced chemiluminescent (ECL) detection method (Amersham,Arlington Heights, Ill.) was used. The lot is exposed to X-ray filmα-PrP RO73 rabbit antiserum is used at a final dilution of 1:5000.

Example 3 Production and Use of a Syrain Hamster Prion Standard

For the development of a standard for calibration of an assay,recombinant Syrian hamster prion proteins of sequence 90-231 wererefolded into α-helical or β-sheet conformations as described [Mehlhorn,Groth et al. (1996) Biochemistry 35:5528-5537]. PCR (Perkin-Elmer) wasused to amplify the DNA corresponding to different portions of theSyrian hamster prion protein in order to ligate it into E. colisecretion vectors. Several 5′ oligonucleotide primers were synthesizedwith an Mlu I restriction site within the C-terminal coding sequence ofthe STI signal peptide [Lee, Moseley et al. (1983) Infect Immun42:264-268; Picken, Mazaitis et al. (1983) Infect Immun 42:269-275] andthe initial amino acids of the appropriate PrP sequence. One 3′oligonucleotide primer matching the 3′ end of PrP, a stop codon and aBam HI restriction site was used with each of the 5oligonucleotides. ThePCR amplified products were purified, ligated into the vectorspreviously digested with MluI/Bam HI and transformed into DH5a. Clonescontaining the PrP insert were sequenced and transformed into theprotease deficient expression strain 27C7 (ATCC# 55244).

Large scale expression was carried out as described previously for otherproteins using a different medium [Carter, Kelley et al. (1992)Biotechnology 10:163-167]; 500 mL of an overnight culture grown in LBmedium supplemented with ampicillin was inoculated into 7 L offermentation medium in an aerated 10 L fermentor (Braun, model E10).Cells were grown at 37° C. at a high agitation rate, and expression wasinduced by phosphate starvation. After 4 h, a 50% glucose solution wasadded at a rate of 1 mL/min; glucose levels were monitored using aglucose dipstick (Diastix, Miles Inc.). A pH of 7.4 was maintainedthroughout the run by the automated addition of 10% H₂SO₄ or 24% NH₄OH.The final volume was 10 L in which an OD₆₀₀ of a ≧100 was achieved after36 h. The E. coli was harvested by centrifugation at 10,000×g for 30 minand the resulting paste was stored at −20° C.

For purification, 100 g of E. coli paste was resuspended in 1 L of 25 mMTris-HCI, pH 8.0, 5 mM EDTA (buffer A). This was centrifuged at 10,000×gfor 20 min, and the supernatant containing soluble periplasmic proteinswas discarded. The pellet was resuspended in 1 L of buffer A, passedthrough a cell disrupter twice (Microfluidics International, modelMF110), and centrifuged at 30,000×g for 1 h, after which the supernatantwas discarded and the pellet was washed once in buffer A and centrifugedagain at 30,000×g for 1 h. At this stage the pellet could be stored at−20° C. prior to further separation. It was subsequently solubilized in8M Gdn-HCI/25 mM Tris-HCI, pH 8.0/100 mM DTT (buffer B) and centrifugedat 14,000×g for 20 min to remove the remaining insoluble matter.Aliquots of 6 mL of the supernatant containing ˜200 mg total proteinwere separated by size exclusion chromatography (SEC) using a 26 mm×60cm HiLoad Superdex 200 column (Pharmacia), eluting with 6M Gdn-HCI/12.5mM Tris-HCI, pH 8.0/5 mM DTT/1 mM EDTA (buffer C) at a flow rate of 2mL/min. Fractions enriched for the recombinant prion protein asidentified by SDS-PAGE were pooled and further purified by reversedphase high performance liquid chromatography (RP-HPLC) employing a 25mm×25 cm C-4 column (Vydac); Buffer 1: H₂O/0.1% TFA, Buffer 2:acetonitrile/0.09% TFA, flow rate 5 mL/min. The recombinant protein rPrPwas found in fractions containing 40% acetonitrile. If the SEC eluatewas stored at 4° C. for several days prior to RP-HPLC, the recombinantprotein was eluted in earlier fractions containing only 35%acetonitrile.

Samples of the reduced protein and the refolded oxidized form wereconcentrated using a Centricon column (Amicon) with a molecular weightcut-off of 10,000 Da. The buffer for the reduced protein was 10 mM MES,pH 6.5 whereas the oxidized form was concentrated in the refoldingbuffer described above. The conformations of refolded oxidized andreduced forms of SHaPrP90-231 protein were determined by circulardichroism (CD) spectroscopy (FIG. 1).

Purified recombinant SHaPrP90-23 T, refolded into α-helical or β-sheetconformation, was diluted into 5% (w/v) brain homogenate obtained fromPrP^(0/0) mouse and containing no prion protein. The brain homogenatewas made by three 30 sec bursts in PowerGen homogenizer equipped withplastic disposable probe in TBS, pH 7.4 containing protease inhibitorscocktail (1 mM PMSF, 2 μg/ml of Aprotinin, and 2 μg/ml of Leupeptin) andspun at 5° C. for 5 min at 500 G in a desktop centrifuge. The resultingsupernatant was diluted 1:1 in TBS with final 4% (w/v) Sarcosyl andhomogenized again by three 30 sec bursts in a PowerGen homogenizer.Next, the homogenate was spiked with different dilutions of recombinantSHaPrP90-231 in α-helical or β-sheet conformations.

In a typical competitive assay, the analyte PrP in differentconfornations is preincubated with europium labeled 3F4 IgG and thentransferred to the polystyrene plate coated with recombinantShaPrP90-231 in SDS-denatured state. The results for analyteSHaPrP90-231 in α-helical and denatured state indicate marked differencein both available binding sites and affinity of europium-labeled 3F4 IgGwith different conformations of prion protein.

In a direct assay, each sample to be tested and the standard weredivided into two aliquots: (1) untreated and designated native; (2)mixed with final 4M Gdn HCI and heated for 5 min at 100° C. anddesignated denatured. Both samples and standard were diluted 20-fold byH₂O and aliquots loaded on polystyrene plate activated withglutaraldehyde. Me plates, incubated overnight at 5° C., were blockedwith TBS, pH 7.8, containing 0.5% BSA (w/v) and 6% Sorbitol (w/v). Inthe next step, they were washed three time with TBS, pH 7.8 containing0.05% (w/v) of Tween® 20 and incubated with europium-labeled antibodieslisted above. The plates were developed after an additional 7 washingsteps in enhancement solution provided by the europium label supplier(Wallac Inc., Turku, Finland) and signal counted on DELFIA 1234Fluorometer (Wallac Inc., Turku, Finland).

Example 4 Creation of a Human Prion Protein Standard and Use inCalibration of Assays

Tg(HuPrP)/PrP^(0/0) mice are created and propagated as described inExample 2. These mice are inoculated intracerebrally with 30 μl ofinfected brain extract using a 27 gauge needle inserted into the rightparietal lobe. The inocula of the mice may be from any human prionpreparation, with examples of such inocula listed below in Table 1. Theincubation time will vary depending on the strain of prion used. See G.C. Telling et al., Cell 83:79-90 (1995) Th following table summarizesexemplary mice strains and inoculum:

Inoculum

(A) Tg(HuPrP)/FVB mice inoculated with sporadic or infectious CJD

-   sCJD(RG)-   sCJD(EC)-   iCJD(364)-   iCJD(364)^(C)-   sCJD(MA)    (B) Tg(HuPrP)/Prnp^(0/0) mice inoculated with sporadic or infectious    CJD-   sCJD(RC)-   sCJD(RG)-   iCJD(364)-   iCJD(364)^(C)-   sCJD(MA)-   sCJD(RO)    (C) Tg(HuPrP)/Prnp^(0/0) mice inoculated with inherited GSS or CJD-   GSS(JJ,P102L)-   fCJD(LJ1,E200K)-   fCJD(CA,E00K)-   fCJD(FH,E200K)

Homogenate of either the brain of a single infected animal or aplurality of animals infected with the same inocula is used to inoculatea larger number of Tg(HuPrP)/PrP^(0/0) mice, which are then followed forsigns of infectivity. Depending on the level of prion protein desired inthe standard, animals can be killed at a specific time followinginnoculation and/or when they exhibit a specific physiological responseto infection, e.g. a certain degree of ataxia. These brain samples arepooled, and a new batch of Tg(HuPrP)/PrP^(0/0) mice inoculated with thehomogenate. This continues, with a new batch of mice used for theproduction of the standard being inoculated with inocula from apreceding generation, and most preferably from the infected micedirectly preceding the new generation. Alternatively, severalgenerations of mice can be infected with the inocula of a single earliergeneration. This procedure allows the standardization of the prionconcentration of the standard while diminishing the background due togenetic variation of the prion preparation.

Human prion proteins are isolated from the mice using techniquesavailable in the art. See e.g. Prusiner et al., Cell 35:349-358 (1983).These proteins can be used to augment a normal human brain preparationfor use as a standard. Total protein concentrations are determined forthe human brain homogenate as described above in Example 2, and then thepreparation can be spiked with the human prion isolates. Spiking a brainhomogenate to create a standard allows the exogenously added form of theprion to be found in a relative concentration to the overall proteinconcentration found in the sample. The sample can then be tested fortotal prion concentration, which would constitute levels of bothendogenous prion concentration and exogenous prion concentration.

Once the standard has been generated and the critical propertiesdetermined, this standard can be used to harmonize data between assays.For example, the comparative prion assay and direct prion assays mayresult in different assay values for a human sample. By performing eachof these assays on the standard with known properties, a correctionvalue may be determined to allow harmonization. The human prion standardis diluted into multiple concentrations: a 1:2 dilution, a 1:5 dilution,a 1:10 dilution and a 1:50 dilution. The competitive and direct assaysare performed on each of the dilutions of the human prion standard. Theresults of the assay values retrieved for each dilution are used todetermine a correction value to harmonize the data to reflect thedetermined true value of prion concentration in the sample.

Example 5 Multimissue Prion Standards

A number of standard brain samples from mice prepared as in Example 3are used in the mold to create a multispecies prion standard for use intesting reagents for specificity and cross-reactivity. Standardizedprion preparations from Tg(SHaPrP)/Prnp^(0/0), Tg(HuPrP)Prnp^(0/0),Tg(ShePrP)Prnp^(0/0), and Tg(BovPrP)/Prnp^(0/0) mice, each infected withthe appropriate strain of prion, are used as the tissue rods in themultitissue preparation. As a control, standardized preparations fromeach strain of transgenic mice not infected with prion can be used as anegative control. In addition, brain samples from physiologically normalhamsters, humans, sheep and cows can be used in the multitissue standardas a further control. Each of these tissues may be removed fromparaffin-blocks, may be fresh tissue or, preferably, the tissue is fixedin any of a variety of tissue fixatives known by those in the art. Foran example of preparation of tissues for this purpose see Battifora, LabInvest 55:244 (1986). Straight rods of tissue of uniform thickness maybe obtained using a multiblade microtome knife.

A tissue embedding mold is created using the techniques as described inBattifora and Mehta, Lab. Invest 63:722-724 (1990). Briefly, the mold isa shallow trough containing parallel ridges separating rectangulargrooves. The prion standard tissue rods are placed within the grooves inthe mold, and 3% agar at a temperature of 60° C. is poured over thetissue rods. The agar is permitted to solidify over a cold plate, andthe gels containing the embedded tissue rods is removed from the mold.The multi-sample block can then be sectioned for use in screeningreagents, testing for antibody specificity, and the like.

The instant invention is shown and described herein in what isconsidered to be the most practical, and preferred embodiments. It isrecognized, however, that departures may be made therefrom, which arewithin the scope of the invention, and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

1. A standardized prion preparation, comprising: prions obtained from a plurality of transgenic mouse brains; and a carrier; wherein the preparation comprises prions (a) which infect and cause disease in an animal chosen from a human, a cow, and a sheep, (b) which are prions of a known strain, (c) the prions are present in a known number of infectious units, and further wherein the carrier is different from brain tissue of the animal chosen from a human, a cow and a sheep; wherein the prions are uniformly dispersed in the preparation and are produced in a transgenic mouse selected from the group consisting of: Tg(HuPrP)/Prnp^(0/0), Tg(ShePrP)/Prnp^(0/0), and Tg(BovPrP)[Prnp^(0/0).
 2. A standardized prion preparation, comprising: prions obtained from a plurality of transgenic mouse brains; and a carrier; wherein the preparation comprises prions (a) which infect and cause disease in a human, (b) which are prions of a known strain, (c) the prions are present in a known number of infectious units, and further wherein the carrier is different from brain tissue of a human; wherein the prions are uniformly dispersed in the preparation and are produced in a transgenic mouse which is Tg(MHu2M)/Prnp^(0/0).
 3. A standardized prion preparation, comprising: prions obtained from a plurality of mice which are Tg (BovPrP)Prnp^(0/0); and a carrier; wherein the prions are present in a known number of infectious units infect and cause disease in a cow and are prions of a known strain. 